A variety of unsymmetrical cyanine dyes and derivatives thereof have been shown to be effective fluorescent stains for nucleic acids. Selected properties of such dyes, such as the brightness, spectral properties, detectability, photostability, affinity for nucleic acids, or selectivity for ss DNA, ds DNA or RNA vary with type and location of dye substituents. Some cyanine dyes are effective stains for reticulocyte analysis (U.S. Pat. No. 4,883,867 to Lee, et al. (1989)) and for bloodborne parasites (U.S. Pat. No.4,937,198 to Lee, et al. (1990)). Other substituted cyanine dyes are typically more fluorescent than the cyanine dyes of Lee et al., and are capable of permeating a variety of cells and electrophoretic gels (U.S. Pat. No. 5,436,134 to Haugland, et al. (1995); U.S. Pat. No. 5,545,535 to Roth et al. (1996); U.S. Pat. No. 5,534,416 to Millard, et al. (1996); and U.S. Pat. No. 5,445,946 to Roth et al. (1995), all incorporated by reference).
Substitution of a cationic side chain makes selected cyanine dyes relatively impermeant to cell membranes (U.S. Pat. No. 5,321,130 to Yue et al. (1994), incorporated by reference), while the substitution of heteroatom-containing side chains also substantially modifies the permeability, selectivity and affinity of the dye for nucleic acids (SUBSTITUTED UNSYMMETRICAL CYANINE DYES WITH SELECTED PERMEABILITY, Ser. No. 08/331,031 by Yue et al., filed Oct. 27, 1994, incorporated by reference).
Chemically reactive compounds have typically been utilized to impart the physical or spectral properties of the compound onto a selected substrate, as in the fluorescent labeling of proteins. Nucleic acid capture reagents comprising methidium have been covalently attached to solid supports, producing materials that bind and subsequently release nucleic acids (U.S. Pat. No. 4,921,805 to Gebeyehu et al. (1990)). Similarly, the attachment of a reactive ester of a carboxylic acid to selected cyanine dyes allows them to be tethered to oligonucleotides, and thereby detect the binding of a target oligonucleotide (U.S. Pat. No. 5,597,696 to Linn et al. (1997)).
The present invention describes chemically reactive versions of cyanine dyes. The invention also describes conjugates of the cyanine dyes prepared using the chemically reactive dyes, and the complex of the dyes of the invention with nucleic acids. Cyanine dyes that are photoreactive possess substantial utility for controlled labeling of biological materials, including nucleic acids. The dye-conjugates of the invention possess utility for the analysis of interactions between proteins and other substrates with nucleic acids, or for immobilizing nucleic acids.